The invention relates to monitoring of phosphorylation or dephosphorylation of a protein.
The post-translational modification of proteins has been known for over 40 years and since then has become a ubiquitous feature of protein structure. The addition of biochemical groups to translated polypeptides has wide-ranging effects on protein stability, protein secondary/tertiary structure, enzyme activity and in more general terms on the regulated homeostasis of cells. Such additions include, but are not limited to, protein phosphorylation and dephosphorylation.
Phosphorylation is a well-studied example of a post-translational modification of proteins. There are many cases in which polypeptides form higher order tertiary structures with like polypeptides (homo-oligomers) or with unalike polypeptides (hetero-oligomers). In the simplest scenario, two identical polypeptides associate to form an active homodimer. An example of this type of association is the natural association of myosin II molecules in the assembly of myosin into filaments.
The dimerization of myosin II monomers is the initial step in seeding myosin filaments. The initial dimerization is regulated by phosphorylation, the effect of which is to induce a conformational change in myosin II secondary structure resulting in the folded 10S monomer subunit extending to a 6S molecule. This active molecule is able to dimerize and subsequently to form filaments. The involvement of phosphorylation of myosin II in this priming event is somewhat controversial. Although in higher eukaryotes the conformational change is dependant on phosphorylation, in Ancanthoamoeba, a lower eukaryote, the post-translational addition of phosphate is not required to effect the initial dimerization step. It is of note that the dimerization domains in myosin II of higher eukaryotes contain the sites for phosphorylation and it is probable that phosphorylation in this region is responsible for enabling myosin II to dimerize and subsequently form filaments. In Dictyostelium this situation is reversed in that the phosphorylation sites are outside the dimerization domain and phosphorylation at these sites is required to effect the disassembly of myosin filaments. In contrast to both these examples, Acanthoamoeba myosin II is phosphorylated in the dimerization domain but this modification is not necessary to enable myosin II monomers to dimerize in this species.
By far the most frequent example of post-translational modification is the addition of phosphate to polypeptides by specific enzymes known as protein kinases. These enzymes have been identified as important regulators of the state of phosphorylation of target proteins and have been implicated as major players in regulating cellular physiology. For example, the cell-division-cycle of the eukaryotic cell is primarily regulated by the state of phosphorylation of specific proteins, the functional state of which is determined by whether or not the protein is phosphorylated. This is determined by the relative activity of protein kinases which add phosphate and protein phosphatases which remove the phosphate moiety from these proteins. Clearly dysfunction of either the kinases or phosphatases may lead to a diseased state. This is best exemplified by the uncontrolled cellular division shown by tumor cells. The regulatory pathway is composed of a large number of genes that interact in vivo to regulate the phosphorylation cascade that ultimately determines if a cell is to divide or arrest cell division.
Currently there are several approaches to analysing the state of modification of target proteins in vivo:
1. In vivo incorporation of labeled (for example, radiolabeled) phosphate, which is added to target proteins. According to one common procedure, intracellular ATP pools are labeled with 32PO4, which is subsequently incorporated into protein. Analysis of modified proteins is typically performed by electrophoresis and autoradiography, with specificity enhanced by immunoprecipitation of proteins of interest prior to electrophoresis.
2. Back-labeling. The incorporation of a labeled phosphate (e.g., 32P) into a protein in vitro to estimate the state of modification in vivo.
3. The use of cell-membrane-permeable protein kinase inhibitors (e.g., Wortmannin, staurosporine) to block phosphorylation of target proteins.
4. Western blotting, of either 1- or 2-dimensional gels bearing test protein samples, in which phosphorylation is detected using antibodies specific for phosphorylated forms of target proteins.
5. The exploitation of eukaryotic microbial systems to identify mutations in protein kinases and/or protein phosphatases.
These strategies have certain limitations. Monitoring states of phosphorylation by pulse or steady-state labeling is merely a descriptive strategy to show which proteins are phosphorylated when samples are separated by gel electrophoresis and visualized by autoradiography. This is unsatisfactory, due to the inability to identify many of the proteins that are phosphorylated. A degree of specificity is afforded to this technique if it is combined with immunoprecipitation; however, this is of course limited by the availability of antibodies to target proteins. Moreover, only highly-expressed proteins are readily detectable using this technique, which may fail to identify many low-abundance proteins, which are potentially important regulators of cellular functions.
The use of kinase inhibitors to block activity is also problematic. For example, very few kinase inhibitors have adequate specificity to allow for the unequivocal correlation of a given kinase with a specific kinase reaction. Indeed, many inhibitors have a broad inhibitory range. For example, staurosporine is a potent inhibitor of phospholipid/Ca+2 dependant kinases. Wortmannin is some what more specific, being limited to the phosphatidylinositol-3 kinase family. This is clearly unsatisfactory because more than one biochemical pathway may be affected during treatment making the assignment of the effects almost impossible.
Monoclonal antibodies directed against phosphorylated epitopes, except in specific cases, exhibit a limitation of specificity comparable to that observed when in vivo labeling is undertaken. Immunological methods can only detect phosphorylated proteins globally (e.g., an anti-phosphotyrosine antibody will detect all tyrosine-phosphorylated proteins) and can only describe a steady state, rather provide a real-time assessment of protein:protein interactions. Such assays also require considerable manpower for processing.
Finally, yeast (Saccharomyces cervisiae and Schizosaccharomyces pombe) has been exploited as a model organism for the identification of gene function using recessive mutations. It is through research on the effects of these mutations that the functional specificities of many protein kinases have been elucidated. However, these molecular genetic techniques are not easily transferable to higher eukaryotes, which are diploid and therefore not as genetically tractable as these lower eukaryotes.
Recent research into the sites of protein phosphorylation has revealed a number of sequence specific motifs which, when phosphorylated or dephosphorylated, promote interaction with selected target proteins to either induce or inhibit activity of either the phosphorylated polypeptide or the target polypeptide.
For example, and not by way of limitation, many proteins involved in intracellular signal transduction have been shown to contain a domain comprising a sequence of approximately 100 amino acids; this sequence is termed the Src homology two (SH2) domain. SH2 domains bind target polypeptides that contain phosphorylated tyrosine. This binding is dependent on the primary amino acid sequence around the phosphotyrosine in the target protein and several peptide sequences which, when phosphorylated, bind to an SH2 domain have been identified (see e.g., Songyang et al., 1993 Cell, 72: 767-778). Non-limiting examples of such sequences include FLPVPEYINQSV (SEQ ID NO: 1), a sequence found in human ECF receptor, and AVGNPEYLNTVQ (SEQ ID NO: 2), a sequence found in human EGF receptor, both of which are autophosphorylated growth factor receptors which stimulate the biochemical signaling pathways that control gene expression, cytoskeletal architecture and cell metabolism. Both of these sequences interact with SH2 domains found in the Sen-5 adapter protein.
The tumor suppressor protein p53, becomes activated by a transcription factor in response to DNA damage. A DNA-dependent protein kinase (DNA-PK) that is activated in response to breaks in DNA is thought to be regulator of p53 activity (Woo et al., 1998, Nature, 394: 700-704). The data described by Woo et al. indicate that the phosphorylation of p53 by DNA-PK serves a dual purpose insofar as phosphorylation promotes the binding of p53 to DNA and also prevents p53 inactivation by MDM2. A p53-derived peptide sequence EPPLSQEAFADLWKK((SEQ ID NO: 3) is identified as the site of phosphorylation in p53 that (when phosphorylated) prevents the interaction of p53 with MDM2.
An example of heterodimer association is described in patent application number WO92/00388. It describes an adenosine 3:5 cyclic monophosphate (cAMP) dependent protein kinase which is a four-subunit enzyme being composed of two catalytic polypeptides (C) and two regulatory polypeptides (R). In nature the polypeptides associate in a stoichiometry of R2C2. In the absence of cAMP the R and C subunits associate and the enzyme complex is inactive. In the presence of cAMP the R subunit functions as a ligand for cAMP resulting in dissociation of the complex and the release of active protein kinase. The invention described in WO92/00388 exploits this association by adding fluorochromes to the R and C subunits.
The polypeptides are labeled (or xe2x80x98taggedxe2x80x99) with fluorophores having different excitation/emission wavelengths. The excitation and emission of one such fluorophore effects a second excitation/emission event in the second fluorophore. By monitoring the fluorescence emission of each fluorophore, which reflects the presence or absence of fluorescence energy transfer between the two, it is possible to derive the concentration of cAMP as a function of the level of association between the R and C. Therefore, the natural affinity of the C subunit for the R subunit has been exploited to monitor the concentration of a specific metabolite, namely cAMP.
The prior art teaches that intact, fluorophore-labeled proteins can function as reporter molecules for monitoring the formation of multi-subunit complexes from protein monomers; however, in each case, the technique relies on the natural ability of the protein monomers to associate.
Tsien et al. (WO97/28261) teach that fluorescent proteins having the proper emission and excitation spectra that are brought into physically close proximity with one another can exhibit fluorescence resonance energy transfer (xe2x80x9cFRETxe2x80x9d). The invention of WO97/28261 takes advantage of that discovery to provide tandem fluorescent protein constructs in which two fluorescent protein labels capable of exhibiting FRET are coupled through a linker to form a tandem construct. In the assays of Tsien et al., protease activity is monitored using FRET to determine the distance between fluorophores controlled by a peptide linker and subsequent hydrolysis thereof. Other applications rely on a change in the intrinsic fluorescence of the protein as in the kinase assays of WO98/06737.
The present invention instead encompasses monitoring of the association of polypeptides, as described herein, which are labeled with fluorescent (protein and chemical) or other labels. FRET, a non-limiting example of a detection method of use in the invention, indicates the proximity of two labeled polypeptide binding partners, which labeled partners associate either in the presence or absence of post-translational addition/removal of a phosphate group to/from a natural binding domain present in at least one of the partners, but not into the fluorophore, reflecting the phosphorylation state of one or both of the binding partners and, consequently, the level of activity of a protein kinase or phosphatase.
There is a need in the art for efficient means of monitoring and/or modulating post-translational protein phosphorylation and/or dephosphorylation. Further, there is a need to develop a technique whereby the addition/removal of a phosphate group can be monitored continuously during real time to provide a dynamic assay system that also has the ability to resolve spatial information.
The invention provides natural binding domains, sequences and polypeptides, as well as kits comprising these molecules and assays of enzymatic function in which they are employed as reporter molecules. As used herein in reference to a polypeptide component of assays of the invention, the term xe2x80x9cnaturalxe2x80x9d refers both to the existence of such an amino acid sequence, whether contiguous or non-contiguous, in nature as well as to the phosphorylation-dependent binding of that component to a second polypeptide or binding partner, and does not relate to attributes of such a polypeptide other than such binding.
One aspect of the invention is an isolated natural binding domain and a binding partner therefor, wherein the isolated natural binding domain includes a site for post-translational phosphorylation and binds the binding partner in a manner dependent upon phosphorylation or dephosphorylation of the site.
The invention also provides a method for monitoring activity of an enzyme comprising performing a detection step to detect binding of an isolated natural binding domain and a binding partner therefor as a result of contacting one or both of the isolated natural binding domain and the binding partner with the enzyme, wherein the isolated natural binding domain includes a site for post-translational phosphorylation and binds the binding partner in a manner dependent upon phosphorylation of the site and wherein detection of binding of the isolated natural binding domain and the binding partner as a result of the contacting is indicative of enzyme activity.
An enzyme to be assayed according to the invention is a protein kinase or a phosphatase.
The invention additionally encompasses a method for monitoring activity of an enzyme comprising performing a detection step to detect dissociation of an isolated natural binding domain from a binding partner therefor as a result of contacting one or both of the isolated natural binding domain and the binding partner with the enzyme, wherein the isolated natural binding domain includes a site for post-translational phosphorylation and binds the binding partner in a manner dependent upon phosphorylation of the site and wherein detection of dissociation of the isolated natural binding domain from the binding partner as a result of the contacting is indicative of enzyme activity.
As used herein, the term xe2x80x9cbinding domainxe2x80x9d in a three-dimensional sense refers to the amino acid residues of a first polypeptide required for phosphorylation-dependent binding between the first polypeptide and its binding partner. The amino acids of a xe2x80x9cbinding domainxe2x80x9d may be either contiguous or non-contiguous and may form a binding pocket for phosphorylation-dependent binding. A domain must include at least 1 amino acid, but may include 2 or more amino acids, preferably at least 4 amino acids, which are contiguous or non-contiguous, but are necessary for phosphorylation-dependent binding to the binding partner. A binding domain will not include a natural full-length polypeptide, but will include a subset of the amino acids of a full-length polypeptide, wherein the subset may include a number of amino acids as high as one fewer than the length of a given natural full-length polypeptide.
A binding domain which is of use in the invention is a xe2x80x9cnatural binding domainxe2x80x9d (i.e., a binding domain that exhibits phosphorylation-dependent binding to a binding partner in nature). A natural binding domain of use in the invention may be isolated or may be present in the context of a larger polypeptide molecule (i.e., one which comprises amino acids other than those of the natural binding domain), which molecule may be either naturally-occurring or recombinant and, in the case of the latter, may comprise either natural or non-natural amino acid sequences outside the binding domain.
As used herein with regard to phosphorylation or dephosphorylation of a polypeptide, the term xe2x80x9csitexe2x80x9d refers to an amino acid or amino acid sequence of a natural binding domain or a binding partner which is recognized by (i.e., a signal for) a kinase or phosphatase for the purpose of phosphorylation or dephosphorylation (i.e., addition or removal of a phosphate moiety) of the polypeptide or a portion thereof. A xe2x80x9csitexe2x80x9d additionally refers to the single amino acid which is phosphorylated or dephosphorylated. It is contemplated that a site comprises a small number of amino acids, as few as one but typically from 2 to 10, less often up to 30 amino acids, and further that a site comprises fewer than the total number of amino acids present in the polypeptide.
In an enzymatic assay of the invention, a xe2x80x9csitexe2x80x9d, for post-translational phosphorylation or dephosphorylation may be present on either or both of the isolated natural binding domain or the binding partner therefor. If such sites are present on both the isolated natural binding domain and its binding partner, binding between the natural binding domain and the binding partner, or between two natural binding domains, may be dependent upon the phosphorylation or dephosphorylation state of either one or both sites. If a single polypeptide chain comprises the natural binding domain and the binding partner (or two natural binding domains), the state of phosphorylation or dephosphorylation of one or both sites will determine whether binding occurs.
A site suitable for addition or removal of a phosphate moiety is present within an isolated natural binding domain or binding partner thereof of the invention at a position such that formation of a complex between the isolated natural binding domain and its binding partner is dependent upon the presence or absence of the phosphate moiety; and preferably does not overlap with an amino acid which is part of a fluorescent tag or other detectable label (including, but not limited to, a radioactive label) or quencher.
Similarly, the amino acid that includes a phosphate moiety may be positioned anywhere within the isolated natural binding domain such that binding of the isolated natural binding domain and its binding partner is dependent upon the presence or absence of the phosphate moiety.
As used herein, xe2x80x9cphosphorylationxe2x80x9d and xe2x80x9cdephosphorylationxe2x80x9d refer to the addition or removal of a phosphate moiety to/from a polypeptide, respectively. As used herein, the term xe2x80x9cpost-translational modificationxe2x80x9d refers to the addition or removal of a phosphate moiety and does not refer to other post-translational events which do not involve addition or removal of a phosphate moiety, and thus does not include simple cleavage of the reporter molecule polypeptide backbone by hydrolysis of a peptide bond.
As used herein, the term xe2x80x9cmoietyxe2x80x9d refers to a post-translationally added or removed phosphate (PO4) group; the terms xe2x80x9cmoietyxe2x80x9d and xe2x80x9cgroupxe2x80x9d are used interchangeably.
As used herein, the term xe2x80x9cbinding partnerxe2x80x9d refers to a polypeptide or fragment thereof (a peptide) that binds to a binding domain, sequence or polypeptide, as defined herein, in a manner which is dependent upon the state of phosphorylation of a site for phosphorylation or dephosphorylation which is, at a minimum, present upon the binding domain, sequence or polypeptide; the binding partner itself may, optionally, comprise such a site and binding between the binding domain, fragment or polypeptide with its corresponding binding partner may, optionally, depend upon modification of that site. A binding partner does not necessarily have to contain a site for phosphorylation or dephosphorylation if such an site is not required to be present on it for modification-dependent association between it and a binding domain, sequence or polypeptide. Binding partners of use in the invention are those which are found in nature and exhibit natural phosphorylation-dependent binding to a natural binding domain, sequence or polypeptide of the invention as defined herein. In one embodiment of the invention, a binding partner is shorter (i.e., by at least one N-terminal or C-terminal amino acid) than the natural full-length polypeptide.
As used herein, the term xe2x80x9cassociatesxe2x80x9d or xe2x80x9cbindsxe2x80x9d refers to a natural binding domain as described herein and its binding partner, having a binding constant sufficiently strong to allow detection of binding by FRET or other detection means, which are in physical contact with each other and have a dissociation constant (Kd) of about 10 xcexcM or lower. The contact region may include all or parts of the two molecules. Therefore, the terms xe2x80x9csubstantially dissociatedxe2x80x9d and xe2x80x9cdissociatedxe2x80x9d or xe2x80x9csubstantially unboundxe2x80x9d or xe2x80x9cunboundxe2x80x9d refer to the absence or loss of contact between such regions, such that the binding constant is reduced by an amount which produces a discernable change in a signal compared to the bound state, including a total absence or loss of contact, such that the proteins are completely separated, as well as a partial absence or loss of contact, so that the body of the proteins are no longer in close proximity to each other but may still be tethered together or otherwise loosely attached, and thus have a dissociation constant greater than 10 xcexcM (Kd). In many cases, the Kd will be in the mM range. The terms xe2x80x9ccomplexxe2x80x9d, xe2x80x9cdimerxe2x80x9d, xe2x80x9cmultimerxe2x80x9d and xe2x80x9coligomerxe2x80x9d as used herein, refer to the natural binding domain and its binding partner in the associated or bound state. More than one molecule of each of the two or more proteins may be present in a complex, dimer, multimer or oligomer according to the methods of the invention.
As used herein in reference to a natural binding domain or other polypeptide, the term xe2x80x9cisolatedxe2x80x9d refers to a molecule or population of molecules that is substantially pure (i.e., free of contaminating molecules of unlike amino acid sequence).
As used herein in reference to the purity of a molecule or population thereof, the term xe2x80x9csubstantiallyxe2x80x9d refers to that which is at least 50%, preferably 60-75%, more preferably from 80-95% and, most preferably, from 98-100% pure.
xe2x80x9cNaturally-occurringxe2x80x9d as used herein, as applied to a polypeptide or polynucleotide, refers to the fact that the polypeptide or polynucleotide can be found in nature. One such example is a polypeptide or polynucleotide sequence that is present in an organism (including a virus) that can be isolated form a source in nature.
The term xe2x80x9csyntheticxe2x80x9d, as used herein, is defined as any amino- or nucleic acid sequence which is produced via chemical synthesis.
In an assay of the invention, post-translational phosphorylation is reversible, such that repeating cycles of addition and removal of a phosphate moiety may be observed, although such cycles may not occur in a living cell found in nature.
An advantage of assays of the invention is that they may, if desired, be performed in xe2x80x9creal timexe2x80x9d. As used herein in reference to monitoring, measurements or observations in assays of the invention, the term xe2x80x9creal timexe2x80x9d refers to that which is performed contemporaneously with the monitored, measured or observed events and which yields a result of the monitoring, measurement or observation to one who performs it simultaneously, or effectively so, with the occurrence of a monitored, measured or observed event. Thus, a xe2x80x9creal timexe2x80x9d assay or measurement contains not only the measured and quantitated result, such as fluorescence, but expresses this in real time, that is, in hours, minutes, seconds, milliseconds, nanoseconds, picoseconds, etc. Shorter times exceed the instrumentation capability; further, resolution is also limited by the folding and binding kinetics of polypeptides.
As used herein, the term xe2x80x9cbinding sequencexe2x80x9d refers to that portion of a polypeptide comprising at least 1, preferably at least 2, more preferably at least 4, and up to 8, 10, 100 or even 1000 contiguous (i.e., covalently linked by peptide bonds) amino acid residues, that are sufficient for phosphorylation-dependent binding to a binding partner. A binding sequence will not include a natural full-length polypeptide, but will include a subset of the amino acids of a full-length polypeptide, wherein the subset may include a number of amino acids as high as one fewer than the length of a given natural full-length polypeptide.
As used herein in reference to those binding sequences that are of use in the invention, the term xe2x80x9cnatural binding sequencexe2x80x9d refers to a binding sequence, as defined above, which consists of an amino acid sequence which is found in nature and which is naturally dependent upon the phosphorylation state of a site for post-translational phosphorylation found within it for binding to a binding partner. A xe2x80x9cnatural binding sequencexe2x80x9d may be present either in isolation or in the context of a larger polypeptide molecule, which molecule may be naturally-occurring or recombinant. If present, amino acids outside of the binding sequence may be either natural, i.e., from the same polypeptide sequence from which the fragment is derived, or non-natural, i.e., from another (different) polypeptide or from a sequence that is not derived from any known polypeptide. In assays of the invention, a binding sequence and its binding partner may exist either on two different polypeptide chains or on a single polypeptide chain.
As used herein, the term xe2x80x9cbinding polypeptidexe2x80x9d refers to a molecule comprising multiple binding sequences, as defined above. A binding polypeptide of use in the invention is a xe2x80x9cnatural binding polypeptidexe2x80x9d, in which the component binding sequences are natural binding sequences, as defined above (e.g., wherein the binding sequences are derived from a single, naturally-occurring polypeptide molecule), and are both necessary and, in combination, sufficient to permit phosphorylation state-dependent binding of the binding polypeptide to its binding partner, wherein the sequences of the binding polypeptide are either contiguous or are non-contiguous. As used herein in reference to the component binding sequences of a binding polypeptide, the term xe2x80x9cnon-contiguousxe2x80x9d refers to binding sequences which are linked by intervening naturally-occurring, as defined herein, or non-natural amino acid sequences or other chemical or biological linker molecules such are known in the art. The amino acids of a polypeptide that do not significantly contribute to the natural phosphorylation-state-dependent binding of that polypeptide to its binding partner may be those amino acids which are naturally present and link the binding sequences in a binding polypeptide or they may be derived from a different natural polypeptide or may be wholly unknown in nature. In assays of the invention, a binding polypeptide and its binding partner (which may, itself, be a binding domain, sequence or polypeptide, as defined herein) may exist on two different polypeptide chains or on a single polypeptide chain. According to the invention, a natural binding polypeptide, like a polypeptide as defined above, is not a full-length natural polypeptide chain, but instead comprises a subset that encompasses up to one fewer than the total number of amino acids in a natural polypeptide chain.
As used herein, the terms xe2x80x9cpolypeptidexe2x80x9d and xe2x80x9cpeptidexe2x80x9d refer to a polymer in which the monomers are amino acids and are joined together through peptide or disulfide bonds. The terms subunit and domain also may refer to polypeptides and peptides having biological function. A peptide useful in the invention will at least have a binding capability, i.e, with respect to binding as- or to a binding partner, and also may have another biological function that is a biological function of a protein or domain from which the peptide sequence is derived. xe2x80x9cPolypeptidexe2x80x9d refers to a naturally-occurring amino acid chain comprising a subset of the amino acids of a full-length protein, wherein the subset comprises at least one fewer amino acid than does the full-length protein, or a xe2x80x9cfragment thereofxe2x80x9d or xe2x80x9cpeptidexe2x80x9d, such as a selected region of the polypeptide that is of interest in a binding assay and for which a binding partner is known or determinable. xe2x80x9cFragment thereofxe2x80x9d thus refers to an amino acid sequence that is a portion of a full-length polypeptide, between about 8 and about 1000 amino acids in length, preferably about 8 to about 300, more preferably about 8 to about 200 amino acids, and even more preferably about 10 to about 50 or 100 amino acids in length. xe2x80x9cPeptidexe2x80x9d refers to a short amino acid sequence that is 10-40 amino acids long, preferably 10-35 amino acids. Additionally, unnatural amino acids, for example, xcex2-alanine, phenyl glycine and homoarginine may be included. Commonly-encountered amino acids which are not gene-encoded may also be used in the present invention. All of the amino acids used in the present invention may be either the D- or L- optical isomer. The L-isomers are preferred. In addition, other peptidomimetics are also useful, e.g. in linker sequences of polypeptides of the present invention (see Spatola, 1983, in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Weinstein, ed., Marcel Dekker, New York, p. 267).
As used herein, the terms xe2x80x9cproteinxe2x80x9d, xe2x80x9csubunitxe2x80x9d and xe2x80x9cdomainxe2x80x9d refer to a linear sequence of amino acids which exhibits biological function. This linear sequence does not include full-length amino acid sequences (e.g. those encoded by a full-length gene or polynucleotide), but does include a portion or fragment thereof, provided the biological function is maintained by that portion or fragment. The terms xe2x80x9csubunitxe2x80x9d and xe2x80x9cdomainxe2x80x9d also may refer to polypeptides and peptides having biological function. A peptide useful in the invention will at least have a binding capability, i.e, with respect to binding as or to a binding partner, and also may have another biological function that is a biological function of a protein or domain from which the peptide sequence is derived.
xe2x80x9cPolynucleotidexe2x80x9d refers to a polymeric form of nucleotides of at least 10 bases in length and up to 1,000 bases or even more, either ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA.
Preferably, with regard to the natural binding domain and/or binding partner therefor, phosphorylation or dephosphorylation is performed by an enzyme which is a kinase or a phosphatase, respectively.
It is preferred that phosphorylation of the site prevents binding of the isolated natural binding domain to the binding partner.
As used herein, the term xe2x80x9cpreventsxe2x80x9d refers to a reduction of at least 10%, preferably 20-40%, more preferably 50-75% and, most preferably, 80-100% of binding of the isolated natural binding domain to the binding partner therefor.
Preferably, phosphorylation of the site promotes binding of the isolated natural binding domain to the binding partner.
As used herein with regard to protein:protein binding, the term xe2x80x9cpromotesxe2x80x9d refers to that which causes an increase in binding of the natural binding domain and its binding partner of at least two-fold, preferably 10- to 20-fold, highly preferably 50- to 100-fold, more preferably from 200- to 1000-fold, and, most preferably, from 200 to 10,000-fold.
It is preferred that dephosphorylation of the site prevents binding of the isolated natural binding domain to the binding partner.
It is additionally preferred that dephosphorylation of the site promotes binding of the isolated natural binding domain to the binding partner.
In a preferred embodiment, at least one of the isolated natural binding domain and the binding partner comprises a detectable label.
Preferably, the detectable label emits light.
More preferably, the light is fluorescent.
It is preferred that one of the isolated natural binding domain and the binding partner therefor comprises a quencher for the detectable label. Labels of use in the invention include, but are not limited to, a radioactive label, a fluorescent label and a quencher for either.
A xe2x80x9cfluorescent labelxe2x80x9d, xe2x80x9cfluorescent tagxe2x80x9d or xe2x80x9cfluorescent groupxe2x80x9d refers to either a fluorophore or a fluorescent protein or fluorescent fragment thereof.
xe2x80x9cFluorescent proteinxe2x80x9d refers to any protein which fluoresces when excited with appropriate electromagnetic radiation. This includes a protein whose amino acid sequence is either natural or engineered. A xe2x80x9cfluorescent proteinxe2x80x9d is a full-length fluorescent protein or fluorescent fragment thereof. By the same token, the term xe2x80x9clinkerxe2x80x9d refers to that which is coupled to both the donor and acceptor protein molecules, such as an amino acid sequence joining two natural binding domains or a disulfide bond between two polypeptides.
It is contemplated that with regard to fluorescent labels employed in FRET, the reporter labels are chosen such that the emission wavelength spectrum of one (the xe2x80x9cdonorxe2x80x9d) is within the excitation wavelength spectrum of the other (the xe2x80x9cacceptorxe2x80x9d). With regard to a fluorescent label and a quencher employed in a single-label detection procedure in an assay of the invention, it is additionally contemplated that the fluorophore and quencher are chosen such that the emission wavelength spectrum of the fluorophore is within the absorption spectrum of the quencher, such that when the fluorophore and the quencher with which it is employed are brought into close proximity by binding of the natural binding domain, sequence or polypeptide upon which one is present with the binding partner comprising the other, detection of the fluorescent signal emitted by the fluorophore is reduced by at least 10%, preferably 20-50%, more preferably 70-90% and, most preferably, by 95-100%. A typical quencher reduces detection of a fluorescent signal by approximately 80%.
Another aspect of the invention is a kit comprising an isolated natural binding domain and a binding partner therefor, wherein the isolated natural binding domain includes a site for post-translational phosphorylation and binds the binding partner in a manner dependent upon phosphorylation of the site, and packaging material therefor.
It is preferred that the kit further comprises a buffer which permits phosphorylation-dependent binding of the isolated natural binding domain and the binding partner.
As used herein, the term xe2x80x9cbufferxe2x80x9d refers to a medium which permits activity of the protein kinase or phosphatase used in an assay of the invention, and is typically a low-ionic-strength buffer or other biocompatible solution (e.g., water, containing one or more of physiological salt, such as simple saline, and/or a weak buffer, such as Tris or phosphate, or others as described hereinbelow), a cell culture medium, of which many are known in the art, or a whole or fractionated cell lysate. Such a buffer permits phosphorylation-dependent binding of a natural binding domain of the invention and a binding partner therefor and, preferably, inhibits degradation and maintains biological activity of the reaction components. Inhibitors of degradation, such as protease inhibitors (e.g., pepstatin, leupeptin, etc.) and nuclease inhibitors (e.g., DEPC) are well known in the art. Lastly, an appropriate buffer may comprise a stabilizing substance such as glycerol, sucrose or polyethylene glycol.
As used herein, the term xe2x80x9cphysiological bufferxe2x80x9d refers to a liquid medium that mimics the salt balance and pH of the cytoplasm of a cell or of the extracellular milieu, such that post-translational protein modification reactions and protein:protein binding are permitted to occur in the buffer as they would in vivo.
Preferably, the buffer permits phosphorylation or dephosphorylation of the site by a kinase or a phosphatase, respectively.
In a preferred embodiment, the kit further comprises one or both of a kinase and a phosphatase.
It is preferred that the kit further comprises a substrate for the phosphatase or kinase, the substrate being MgATP.
It is contemplated that at least a part of a substrate of an enzyme of use in an assay of the invention is transferred to a phosphorylation site on an isolated polypeptide of the invention. As used herein, the term xe2x80x9cat least a part of a substratexe2x80x9d refers to a portion (e.g., a moiety or a group, as defined above) which comprises less than the whole of the substrate for the enzyme, the transfer of which portion to a phosphorylation site on an isolated polypeptide, both as defined above, is catalyzed by the enzyme.
It is additionally preferred that the kit further comprises a cofactor for one or both of the kinase or phosphatase. Cofactors of use in the invention include, but are not limited to, cAMP, phosphotidylserine, diolein, Mn2+ and Mg2+.
Preferably, at least one of the isolated natural binding domain and the binding partner comprises a detectable label.
It is preferred that the detectable label emits light, and more preferred that the light is fluorescent.
An enzyme (e.g., a protein kinase or phosphatase) of use in the invention may be natural or recombinant or, alternatively, may be chemically synthesized. If either natural or recombinant, it may be substantially pure (i.e., present in a population of molecules in which it is at least 50% homogeneous), partially purified (i.e., represented by at least 1% of the molecules present in a fraction of a cellular lysate) or may be present in a crude biological sample.
As used herein, the term xe2x80x9csamplexe2x80x9d refers to a collection of inorganic, organic or biochemical molecules which is either found in nature (e.g., in a biological- or other specimen) or in an artificially-constructed grouping, such as agents which might be found and/or mixed in a laboratory. Such a sample may be either heterogeneous or homogeneous.
As used herein, the interchangeable terms xe2x80x9cbiological specimenxe2x80x9d and xe2x80x9cbiological samplexe2x80x9d refer to a whole organism or a subset of its tissues, cells or component parts (e.g. body fluids, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen). xe2x80x9cBiological samplexe2x80x9d further refers to a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof. Lastly, xe2x80x9cbiological samplexe2x80x9d refers to a medium, such as a nutrient broth or gel in which an organism has been propagated, which contains cellular components, such as proteins or nucleic acid molecules.
As used herein, the term xe2x80x9corganismxe2x80x9d refers to all cellular life-forms, such as prokaryotes and eukaryotes, as well as non-cellular, nucleic acid-containing entities, such as bacteriophage and viruses.
In a method as described above, it is preferred that at least one of the isolated natural binding domain and the binding partner is labeled with a detectable label.
Preferably, the label emits light and, more preferably, the light is fluorescent.
In another preferred embodiment, the detection step is to detect a change in signal emission by the detectable label.
It is preferred that the method further comprises exciting the detectable label and monitoring fluorescence emission.
It is additionally preferred that the method further comprises the step, prior to or after the detection step, of contacting the isolated natural binding domain and the binding partner with an agent which modulates the activity of the enzyme.
As used herein with regard to a biological or chemical agent, the term xe2x80x9cmodulatexe2x80x9d refers to enhancing or inhibiting the activity of a protein kinase or phosphatase in an assay of the invention; such modulation may be direct (e.g. including, but not limited to, cleavage of- or competitive binding of another substance to the enzyme) or indirect (e.g. by blocking the initial production or, if required, activation of the kinase or phosphatase).
xe2x80x9cModulationxe2x80x9d refers to the capacity to either increase or decease a measurable functional property of biological activity or process (e.g., enzyme activity or receptor binding) by at least 10%, 15%, 20%, 25%, 50%, 100% or more; such increase or decrease may be contingent on the occurrence of a specific event, such as activation of a signal transduction pathway, and/or may be manifest only in particular cell types.
The term xe2x80x9cmodulatorxe2x80x9d refers to a chemical compound (naturally occurring or non-naturally occurring), such as a biological macromolecule (e.g., nucleic acid, protein, non-peptide, or organic molecule), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues, or even an inorganic element or molecule. Modulators are evaluated for potential activity as inhibitors or activators (directly or indirectly) of a biological process or processes (e.g., agonist, partial antagonist, partial agonist, antagonist, antineoplastic agents, cytotoxic agents, inhibitors of neoplastic transformation or cell proliferation, cell proliferation-promoting agents, and the like) by inclusion in screening assays described herein. The activities (or activity) of a modulator may be known, unknown or partially-known. Such modulators can be screened using the methods described herein.
The term xe2x80x9ccandidate modulatorxe2x80x9d refers to a compound to be tested by one or more screening method(s) of the invention as a putative modulator. Usually, various predetermined concentrations are used for screening such as 0.01 xcexcM, 0.1 xcexcM, 1.0 xcexcM, and 10.0 xcexcM, as described more fully hereinbelow. Test compound controls can include the measurement of a signal in the absence of the test compound or comparison to a compound known to modulate the target.
The invention additionally provides a method of screening for a candidate modulator of enzymatic activity of a kinase or a phosphatase, the method comprising contacting an isolated natural binding domain, a binding partner therefor and an enzyme with a candidate modulator of the kinase or phosphatase, wherein the natural binding domain includes a site for post-translational phosphorylation and binds the binding partner in a manner that is dependent upon phosphorylation or dephosphorylation of the site by the kinase or phosphatase and wherein at least one of the isolated natural binding domain and the binding partner comprises a detectable label, and monitoring the binding of the isolated natural binding domain to the binding partner, wherein binding or dissociation of the isolated natural binding domain and the binding partner as a result of the contacting is indicative of modulation of enzymatic activity by the candidate modulator of the kinase or phosphatase.
Preferably, the detectable label emits light.
More preferably, the light is fluorescent.
It is preferred that the monitoring comprises measuring a change in energy transfer between a detectable label present on the isolated natural binding domain and a detectable label present on the binding partner.
A final aspect of the invention is a method of screening for a candidate modulator of enzymatic activity of a kinase or a phosphatase, the method comprising contacting an assay system with a candidate modulator of enzymatic activity of a kinase or phosphatase, and monitoring binding of an isolated natural binding domain and a binding partner therefor in the assay system, wherein the isolated natural binding domain includes a site for post-translational phosphorylation and binds the binding partner in a manner that is dependent upon phosphorylation or dephosphorylation of the site by a kinase or phosphatase in the assay system, wherein at least one of the isolated natural binding domain and the binding partner comprises a detectable label, and wherein binding or dissociation of the isolated natural binding domain and the binding partner as a result of the contacting is indicative of modulation of enzymatic activity by the candidate modulator of a the kinase or phosphatase.
It is highly preferred that in any of the above methods, the method comprises real-time observation of association of an isolated natural binding domain and its binding partner.